738¨747
`J. Inher. Metab. Dis. 21
`(1998)
`(
`SSIEM and Kluwer Academic Publishers. Printed in the Netherlands
`
`Homocystinuria due to cystathionine b-synthase
`deficiency in Ireland : 25 years(cid:146) experience of a
`newborn screened and treated population with
`reference to clinical outcome and biochemical
`control
`
`S. YAP and E. NAUGHTEN*
`National Centre for Inherited Metabolic Disorders, T he Childrenˇs Hospital, T emple
`Street, Dublin 1, Ireland
`* Correspondence
`
`MS received 27.06.97 Accepted 26.02.98
`
`Summary: Homocystinuria (HCU) due to cystathionine b-synthase de—ciency
`(Mudd et al 1964) was independently described by Gerritsen and colleagues
`(USA) and Carson and colleagues (Northern Ireland) in 1962. The worldwide
`frequency of HCU has been reported as 1 in 344 000, while that in Ireland is
`much higher at 1 in 65 000, based on newborn screening and cases detected
`clinically. The national newborn screening programme for HCU in Ireland was
`started in 1971 using the bacterial inhibition assay. A total of 1.58 million
`newborn infants have been screened over a 25-year period up to 1996. Twenty-
`—ve HCU cases were diagnosed, 21 of whom were identi—ed on screening. The
`remaining four HCU cases were missed and presented clinically; three of these
`were breast-fed and one was pyridoxine responsive. Twenty-four HCU cases
`were pyridoxine nonresponsive. Once the status of pyridoxine responsiveness
`was identi—ed, all pyridoxine nonresponsive cases, but one, were started on a
`low methionine, cystine-enhanced diet supplemented with pyridoxine, vitamin
`B12
`and folate. Dietary treatment commenced within 6 weeks of birth (range
`8¨42 days) for those cases detected by screening, while for the late-detected
`cases treatment was started upon presentation and diagnosis. Biochemical
`control was monitored measuring deproteinized plasma methionine, free homo-
`cystine and cystine at least once a month. Review of the clinical outcome of the
`25 HCU cases with 365.7 patient-years of treatment revealed no HCU-related
`complications in 18 screened, dietary-treated cases. Fifteen of these had lifetime
`medians of free homocystine „11 kmol/L (range 4¨11). The remaining three
`cases with higher lifetime medians of free homocystine (18, 18 and 48 kmol/L)
`have developed increasing myopia recently. Among the three screened non-
`dietary-compliant cases, two have ectopia lentis, one has osteoporosis and two
`have mental handicap. Of the four cases missed on screening, three presented
`
`738
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`
`

`

`Homocystinuria in Ireland
`
`739
`
`with ectopia lentis after the age of 2 years. There were no thromboembolic
`events in any of the 25 HCU cases. The lifetime medians for methionine ranged
`from 47 to 134 kmol/L. The Irish HCU clinical outcome data suggest that
`newborn screening, early commencement of dietary treatment and a lifetime
`median of free homocystine of „ 11 kmol/L had signi—cantly reduced the prob-
`ability of developing complications when it was compared to the untreated
`HCU data (Mudd et al 1985).
`
`(HCU; McKusick 236200) due to cystathionine b-synthase (CBS;
`Homocystinuria
`EC 4.2.1.22) de—ciency is an autosomal recessive disorder with a frequency of 1 in
`65 000 in Ireland, based on newborn screening and cases detected clinically, com-
`pared to a worldwide frequency of 1 in 344 000 (Mudd et al 1995). The national
`newborn screening programme was started in Ireland for HCU in 1971. Approx-
`imately one half of individuals with HCU worldwide are pyridoxine responsive,
`resulting in a decrease in plasma concentration of methionine and almost complete
`elimination of homocyst(e)ine in the blood and urine (Andria and Sebastio, 1996).
`Pyridoxine-nonresponsive cases have a more severe clinical presentation. The CBS
`gene has been mapped to the human chromosome 21q22.3 (Munke et al 1988). In
`Ireland, 70¨71% of the defective CBS alleles are G307S, which heralds a more
`severe and earlier clinical presentation in association with pyridoxine non-
`responsiveness (Gallagher et al 1995).
`Individuals with HCU are clinically normal at birth. If untreated, they may
`present with varying degrees of the well-recognized clinical syndrome of ectopia
`lentis, dolichostenomelia, osteoporosis, thromboembolic events, and mental retar-
`dation. Of untreated patients who are pyridoxine nonresponsive, 82% will have
`ectopia lentis by the age of 10 years, 27% will have had a clinically detected throm-
`boembolic event by the age of 15 years, 64% will have radiological evidence of
`spinal osteoporosis by the age of 15 years, and 23% will not survive to the age of 30
`years (Mudd et al 1985). This paper presents the clinical outcome and biochemical
`control of 25 cases of HCU in Ireland detected in the 25-year period of national
`newborn screening with 365.7 patient-years of treatment. Four of these 25 cases
`were missed on screening.
`
`PATIENTS AND METHODS
`Subjects: This is a retrospective study of all 25 cases of HCU detected in Ireland
`between 1971 and 1996 either by the national newborn screening programme or by
`clinical presentation. The data were extracted from the clinical notes.
`There are 12 females and 13 males from 19 families; one male died at the age of 8
`years following a drowning accident. Twenty-four of the twenty-—ve patients are
`nonresponsive to pyridoxine. Twenty-one cases were identi—ed by the national
`newborn screening programme. Of the remaining four cases missed on screening,
`three were breast-fed and one is pyridoxine responsive. All but one were started on
`treatment upon diagnosis.
`
`J. Inher. Metab. Dis. 21 (1998)
`
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`

`740
`
`Y ap and Naughten
`
`Biochemical determinants: All 21 cases of HCU detected by newborn screening had
`high blood methionine concentration of more than 100 kmol/L, assayed by the bac-
`terial inhibition assay (BIA) on the heel prick blood sample taken on day 3 to day 5
`of life. A liquid blood sample, separated and deproteinized within 10 min of collec-
`tion, was requested from these infants and assayed using an amino acid analyser for
`methionine, free homocystine and cystine. A high blood methionine and free homo-
`cystine with a low cystine con—rmed the diagnosis for those detected on newborn
`screening and those that presented clinically. CBS enzyme activity in skin —bro-
`blasts, assessed by Fowler according to his methods (Fowler et al 1978), was only
`available for 5 patients. Three patients showed no detectable CBS activity in both
`assays with or without added 1.0 mmol/L pyridoxal phosphate (normal CBS activity
`4¨25 nmol/h per mg). The remaining two patients exhibited very minimal CBS
`activity (0.05 and 0.037 increasing to 0.066 and 0.062 nmol/h per mg respectively
`with the addition of 1 mmol/L pyridoxal phosphate).
`
`Clinical management and follow-up: All patients diagnosed as having HCU had
`their pyridoxine status determined clinically by commencement on oral pyridoxine
`50 mg three times daily as inpatients. The deproteinized blood methionine, free
`homocystine and cystine were initially determined every 3 days while on daily oral
`pyridoxine. Pyridoxine responsiveness was indicated by a rapidly falling methionine
`level and clearing of free homocystine from the plasma, while pyridoxine non-
`responsiveness was indicated by persistently high or rising plasma methionine and
`free homocystine.
`In pyridoxine-nonresponsive patients, dietary management was commenced by
`restricting dietary methionine and using a methionine-free, cystine-supplemented
`synthetic mixture. Methionine was added as a proprietary food preparation or as
`breast milk and the amount was titrated against the plasma methionine and free
`homocystine concentration. Two-thirds of the total protein intake was derived from
`the synthetic methionine-free, cystine-supplemented mixture and the remaining third
`from the natural methionine-containing foods (P. Howard, personal communica-
`B12
`tion, 1996). Plasma
`and folate were assayed and, if they were found de—cient, the
`patient was given supplements. Once stabilized, blood was drawn, deproteinized,
`and analysed for methionine, free homocystine and cystine at least once a month, or
`more frequently if clinically indicated, to monitor biochemical control. During all
`episodes of acute illness, prompt treatment of the illness with additional supportive
`care,
`including adequate hydration and aspirin or dipyridamole, was given to
`prevent blood stasis.
`Lifetime medians and ranges of plasma methionine and free homocystine levels
`were calculated. Methionine and free homocystine levels during the initial periods of
`stabilization, pyridoxine challenge and subsequent periods of proven illness were
`excluded from the calculations for lifetime medians and ranges.
`Upon discharge from hospital, patients initially attended the outpatient depart-
`ment fortnightly and thereafter at 4- to 6-weekly intervals. They were reviewed mini-
`mally four times per year. At every clinic visit, growth parameters were measured, a
`general physical examination was carried out by a doctor, and blood was drawn,
`
`J. Inher. Metab. Dis. 21 (1998)
`
`Lilly Ex. 2095
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`
`

`

`Homocystinuria in Ireland
`
`741
`
`deproteinized and analysed for methionine, free homocystine and cystine to monitor
`biochemical control. Experienced dietitians provided dietary assessments and con-
`stant advice at each visit. Annual detailed ophthalmological examination was per-
`formed by a paediatric ophthalmologist experienced in HCU. Initially, skeletal
`radiology was performed yearly to detect evidence of osteoporosis. Up to 1990,
`annual clinical cardiovascular assessments including electrocardiographs and chest
`radiographs were performed by experienced paediatric cardiologists. Transthoracic
`echocardiograms were carried out when clinical abnormalities (e.g. murmurs, pulse
`di(cid:134)erences) were detected. IQ assessments were performed at 2- to 6-yearly intervals.
`Psychological support was provided whenever necessary by the clinical psychol-
`ogist.
`
`RESULTS
`Patient demographics:
`In 25 years of national newborn screening up to 1996, 21
`cases of HCU were detected through the programme. All were pyridoxine non-
`responsive and —ve cases were breast-fed. Four other HCU cases were missed and
`presented clinically; three were breast-fed and one was pyridoxine responsive.
`Details of complications among the study group are categorized in Table 1.
`
`Clinical and biochemical —ndings: The 25 cases of HCU are divided into three
`groups according to the mode of detection (screened and missed on screening) and
`the development of complications. Group 1 consisted of 18 HCU cases detected on
`screening,
`treated and remaining free from the recognized complications of
`untreated HCU (Table 2). Three patients who were detected by screening developed
`complications due to noncompliance with the prescribed diet, as indicated by
`dietary history and poor biochemical control, are in group 2 (Table 3). The four
`cases missed on screening presented with complications after the age of 2 years and
`are in group 3 (Table 4).
`One of the four cases missed on screening and never treated was recently referred
`to our clinic aged 21 years with recognized complications of untreated HCU (case 4,
`Table 4). She had bilateral ectopia lentis and optic atrophy, mental retardation,
`
`Table 1 Complications among the HCU patients
`
`Detected by screening
`
`T otal no.
`
`25
`6
`2
`
`Total no. detected
`Ectopia lentis
`Osteoporosis
`(radiological)
`4
`Mental handicap
`0
`Thromboembolism
`a One died at age 8 years due to a drowning accident.
`
`W ithout
`complications
`18a
`0
`0
`
`0
`0
`
`W ith
`complications
`
`Missed on
`screening
`
`3
`2
`1
`
`2
`0
`
`4
`4
`1
`
`2
`0
`
`J. Inher. Metab. Dis. 21 (1998)
`
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`
`

`

`Y ap and Naughten
`742
`Table 2 Summary of age data, lifetime medians and ranges of free homocystine (lmol/L) and
`methionine (lmol/L) for cases in group 1 (screened, on diet with no complications)
`
`Age
`(1996)
`(years)
`
`Age
`therapy
`started
`(days)
`
`Case
`no.
`
`Sex
`
`L ifetime free homocystine
`
`L ifetime methionine
`
`No.
`
`Median
`
`Range
`
`No.
`
`Median
`
`Range
`
`1
`M
`2.5
`15
`24
`4.0
`2
`M
`4.3
`15
`29
`6.0
`3
`F
`5
`8
`32
`5.5
`4
`F
`6.5
`10
`51
`6.0
`5a
`M
`8
`21
`19
`11
`6b
`F
`12.7
`28
`64
`5.0
`7
`M
`12.8
`22
`68
`11
`8
`M
`13
`7
`41
`8.0
`9
`M
`13.9
`13
`66
`10
`10
`F
`14.6
`15
`85
`6.0
`11
`M
`14.6
`21
`52
`5.5
`12b
`F
`15.9
`26
`66
`18
`13
`F
`17.1
`8
`73
`18
`14
`M
`20.7
`10
`39
`7.5
`15b
`M
`20.9
`10
`60
`8.5
`16
`M
`21.8
`36
`65
`9.0
`17b
`F
`22.5
`35
`75
`9.0
`18b
`M
`23.4
`42
`97
`48
`a Case 5 died at the age of 8 years due to drowning accident
`b Skin —broblast assayed for CBS activity
`M \ Male, F \ female
`
`0¨17
`0¨19
`0¨19
`0¨19
`0¨30
`0¨47
`0¨34
`0¨22
`0¨25
`0¨36
`0¨21
`0¨86
`0¨88
`0¨87
`0¨55
`0¨70
`0¨29
`0¨172
`
`31
`26
`33
`51
`19
`62
`70
`41
`67
`84
`52
`63
`73
`50
`8
`73
`64
`98
`
`56
`56.5
`48
`47
`87.2
`61
`64
`64
`57
`67.4
`63.5
`80
`88
`56.3
`97.5
`134
`96
`99.5
`
`5¨651
`35¨76
`5¨80
`6¨110
`21¨590
`11¨173
`7¨232
`19¨132
`13¨187
`21¨300
`18¨314
`12¨448
`10¨365
`1¨268
`24¨642
`23¨762
`33¨412
`27¨523
`
`osteoporosis, dolichostenomelia (arm span 164 cm to height of 163 cm) but no docu-
`mented clinical evidence of thromboembolic events.
`The individual age at which diet was commenced, lifetime medians and ranges of
`plasma methionine and free homocystine levels are summarized in Tables 2, 3 and 4.
`Treatment was started before 6 weeks of age for patients in groups 1 and 2, while
`those in group 3 started upon presentation and diagnosis. The mean period of
`follow-up for the screened groups 1 and 2 was 14.3 years (range 2.5¨23.4) and for the
`late-detected group 3 was 14.7 years (range 11.7¨18.8).
`Three patients (cases 12, 13 and 18) in group 1 (Table 2) developed increasing
`myopia in the last few years and all had higher lifetime median of plasma free
`homocystine (18, 18 and 48 kmol/L) compared to the remaining 15 cases, who all
`had lifetime medians of plasma free homocystine levels „11 kmol/L (Figure 1). No
`patient, whether detected by screening or clinically, developed any thromboembolic
`events, in contrast to the predicted outcome at their current age according to the
`time-to-event graphs by Mudd et al (1985; see Table 6). The lifetime median methi-
`onine levels ranged from 47 to 134 kmol/L.
`Four patients had mental handicap: two noncompliant patients detected by
`screening (group 2: cases 1 and 2) and two who were missed on screening (group 3:
`cases 1 and 4). The remaining 21 patients have achieved age-appropriate education
`standards (Table 5).
`
`J. Inher. Metab. Dis. 21 (1998)
`
`Lilly Ex. 2095
`Sandoz v. Lilly IPR2016-00318
`
`

`

`Homocystinuria in Ireland
`
`743
`
`Complications
`Osteoporosisa
`BIQ
`Ectopia lentis
`BIQ
`Iridodenesis
`
`Iridodenesis
`
`13
`
`42
`
`97
`
`83
`
`49
`
`18
`
`0¨108
`
`0¨81
`
`101
`
`79
`
`70
`
`80
`
`4¨405
`
`4¨789
`
`9
`Compliant
`Noncompliant
`
`55
`
`11
`5.5
`58
`
`0¨96
`0¨96
`24¨71
`
`63
`52
`11
`
`80
`81
`85
`
`0¨536
`0¨536
`43¨161
`
`Table 3 Dietary noncompliant screened patients (group 2) with corresponding complications, lifetime medians and ranges of
`free homocystine (lmol/L) and methionine (lmol/L)
`Age
`1996
`(years)
`
`Age therapy
`started (days)
`
`Free homocystine
`
`Methionine
`
`No.
`
`Median
`
`Range
`
`No. Median
`
`Range
`
`Case
`no./Sex
`
`1/female
`
`12
`
`16.5
`
`2/female
`3/femaleb
`
`J. Inher. Metab. Dis. 21 (1998)
`
`22.8
`1¨15
`16¨22.5
`a Radiological osteoporosis
`b Case 3 had a free homocystine median of 5.5 kmol/L in the —rst 15 years of life when she was complying with dietary treatment (italic). The
`free homocystine median increased to 58 kmol/L in the following 6.5 years when she was noncompliant with the diet and presented with
`iridodenesis (italic). Her overall lifetime (22.8 years) median of free homocystine was 11 kmol/L
`
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`
`

`

`Y ap and Naughten
`744
`Table 4 Summary of age data, lifetime medians and ranges of free homocystine (lmol/L) and
`methionine (lmol/L) for cases in group 3 (missed on screening, presenting with complications)
`Age
`therapy
`started
`(years)
`
`L ifetime free homocystine
`
`L ifetime methionine
`
`No. Median Range No. Median Range
`0¨38
`23¨153
`0¨62
`3¨791
`0¨27
`36¨116
`¨
`¨
`
`56
`94
`30
`¨
`
`4.5
`8.5
`5.0
`¨
`
`56
`82
`30
`¨
`
`50
`67
`64
`¨
`
`Age
`(1996)
`(years)
`
`16.8
`17
`18.7
`20.8
`
`2.4
`2.9
`7.0
`¨
`
`nonresponsive
`
`Case
`Sex
`no.
`1a Male
`2a
`Female
`3b Male
`4a
`Female
`a Breast-fed,
`B6
`b B6
`responsive
`Cases 2, 3 and 4 presented with ectopia lentis
`
`DISCUSSION
`
`Newborn screening and early dietary intervention signi—cantly reduce the morbidity
`and mortality in patients with HCU. Although the number of cases studied in this
`paper (25) was comparatively small with varying lengths of follow-up, the lack of
`
`Figure 1 Screened HCU patients on diet with no complications. Data points represent
`current age of HCU patients with their corresponding lifetime medians of free homocystine
`(kmol/L) (group 1). Increasing myopia has developed in three patients with the highest life-
`time medians for free homocystine in this group
`
`J. Inher. Metab. Dis. 21 (1998)
`
`Lilly Ex. 2095
`Sandoz v. Lilly IPR2016-00318
`
`

`

`Homocystinuria in Ireland
`
`745
`
`Table 5 Educational achievement of 25 patients with HCU in Ireland
`
`Educational
`attainment
`
`Age
`(years)
`[18
`
`12¨18
`
`Tertiary-level
`education
`Secondary school
`Remedial help
`Special school
`5¨11
`Primary school
`\5
`Pre-school
`a One died due to drowning accident
`
`T otal
`no.
`
`Screened
`compliant
`
`Screened
`noncompliant
`
`Missed on
`screening
`
`7
`
`6
`2
`1
`8a
`4
`
`4/4
`
`2/2
`
`7/7
`4/4
`
`3/3
`2
`
`1/3
`
`1/1
`
`1
`
`obvious clinical complications was very encouraging. When the Irish outcome data
`were compared to that of Mudd et al (1985), there was a notable reduction in the
`probability of developing complications (Table 6), in particular the development of
`thromboembolic events. Poor dietary compliance, as re(cid:209)ected by dietary history and
`higher plasma free homocystine levels for between 2 and 8 years, signi—cantly
`increased the risk of developing complications. Compliance with diet for life can be
`a problem at all ages, particularly during adolescence, despite the possible bene—cial
`e(cid:134)ects in preventing complications (Brenton et al 1966; Parkinson 1969). It does,
`however, seem achievable with continuous support for the patient and family from a
`dedicated multidisciplinary team. Betaine, a methyl donor, may be needed to
`
`Table 6 Probability of not developing clinical complications in the 18 Irish HCU patients at
`their current ages, if they were untreated, based on the data of Mudd et al (1985)
`
`Current age (years)
`
`Ectopia lentis
`
`Osteoporosis
`
`T hromboembolism
`
`Percentage probability of not developing complications
`
`2.5
`4.3
`5.0
`6.5
`8.0
`12.7
`12.8
`13.0
`13.9
`14.6
`14.6
`15.9
`17.1
`20.1
`20.7
`21.8
`22.5
`23.4
`
`96
`64
`53
`43
`13.2
`13.2
`13.2
`8
`8
`
`98
`96
`90
`84
`48
`48
`43
`40
`40
`35
`31
`26
`
`98
`96
`95
`93
`84
`84
`82
`78
`78
`72
`66
`64
`64
`56
`56
`52
`
`J. Inher. Metab. Dis. 21 (1998)
`
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`Sandoz v. Lilly IPR2016-00318
`
`

`

`746
`
`Y ap and Naughten
`
`augment dietary treatment in noncompliant HCU patients or used as the sole treat-
`ment where diet is unacceptable to the patient (Wilcken et al 1983).
`Early detection, good dietary compliance and a lifetime median of plasma free
`homocystine „11 kmol/L seems to protect against the recognized complications as
`demonstrated by 15 cases in group 1. Increasing myopia may be the —rst sign of
`persistent poor compliance, prior to ectopia lentis (Burke et al 1989), as seen in three
`cases in group 1 with high lifetime medians of free homocystine levels of 18, 18 and
`48 kmol/L, despite their insistence on good dietary compliance. All 25 cases of HCU
`had high lifetime median plasma methionine ranging from 47 to 134 kmol/L and
`this hypermethioninaemia does not appear to be toxic. Our data, as provided by
`group 3, also agree with the —ndings of Mudd et al (1985) that (cid:147)there is a lag period
`of approximately two yearsˇ before appreciable lens dislocation occurs in the
`untreated HCU patients. Mudd et al (1985), in a large HCU group, reported that
`the chance of su(cid:134)ering a clinically detected thromboembolic event for the untreated
`pyridoxine-nonresponsive patient is 25% by the age of 16 years and 50% by 29
`years, with the maximal likelihood of such an event occurring between 12.5 and 17.5
`years of age. None of the patients reported in this study developed detectable
`thromboembolic events (age range 2.5¨23.4 years).
`These —ndings show that classical homocystinuria is a potentially treatable
`inherited metabolic disorder. Newborn screening, early treatment, good dietary
`compliance and the maintenance of a lifetime median plasma free homocystine
`„11 kmol/L seem to protect, at least up to 23.4 years, against the overt recognized
`complications of untreated HCU. Additional long-term follow-up of this group of
`HCU patients with more precise and sensitive methods of cardiovascular and bone
`mineralization assessment will provide further vital information for evaluating the
`efficacy of early dietary treatment in preventing complications.
`
`ACKNOWLEDGEMENTS
`
`The
`authors acknowledge the following for their contribution to the diagnosis and
`management of these HCU patients: Dr S. Cahalane and Dr P. D. Mayne, former
`and present directors of the National Newborn Screening Programme in Ireland,
`and the laboratory sta(cid:134); Dr D. Murphy, former consultant paediatrician with a
`special interest in inherited metabolic disorders, The Childrenˇs Hospital, Dublin;
`Dr B. Fowler, head of laboratory, Basler Kinderspital, Switzerland; the nursing,
`dietetic, ophthalmology, cardiology, radiology and psychology colleagues of The
`Childrenˇs Hospital, Dublin; Dr P. Thornton, consultant paediatrician with a
`special interest in inherited metabolic disorders, The Childrenˇs Hospital, Dublin,
`for allowing the inclusion of a recently referred HCU patient (case 4, Table 4) into
`our study group; and Dr M. OˇRegan, senior lecturer, Department of Statistics,
`Trinity College, Dublin.
`
`REFERENCES
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